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Frequency of factors associated with habitual abortion in 197 couples*†
FERTILITY AND STERILITY® Copyright
,t'J
Vol. 66, No.1, July 1996 Printed on acidfree paper in U. S. A.
1996 American Society for Reproductive Medicine
Frequency of factors associated with habitual abortion in 197 couples*t
Mary D. Stephenson, M.D.:!: Department of Obstetrics and Gynaecology, University of British Columbia, British Columbia's Women's Hospital and Health Centre, Vancouver, British Columbia, Canada
Objective: To determine the frequency of factors associated with habitual abortion in 197 couples. Design: Prospective cohort study. Setting: The British Columbia Recurrent Pregnancy Loss Program, located in a tertiary care academic center. Interventions: Diagnostic screening protocol. Main Outcome Measures: Genetic, endocrine, infectious, anatomical, and autoimmune factors associated with habitual abortion. Results: A structural genetic factor was identified in 3.5% ofthe couples. An endocrine factor, including luteal phase deficiency and hypothyroidism, was identified in 20% and an infectious factor was identified in 0.5% of the couples. An anatomical factor, including various miillerian tract anomalies and severe intrauterine adhesions, was found in 16% and an autoimmune factor, including the antiphospholipid antibody syndrome and the undifferentiated connective tissue syndrome, was identified in 20% of the couples. Eighty-four couples who completed the diagnostic screening protocol were classified as having unexplained habitual abortion. Of these 84 couples, 65% were subclassified as primary, 27% were subclassified as secondary, and 7% had unclassified unexplained habitual abortion. Conclusions: This large-scale study identified genetic, endocrine, infectious, anatomical, or autoimmune factors in approximately 60% of couples with habitual abortion. Fertil Steril® 1996;66:24-9 Key Words: Habitual abortion, etiology, factors, frequency
Habitual abortion previously has been defined as three or more consecutive spontaneous abortions (1). The World Health Organization has defined an abortion as "the expulsion or extraction from its mother of an embryo or fetus weighing 500 grams or less" (2), which corresponds to a gestational age between 20 and 22 weeks. It appears that 15% of all clinically Received October 18, 1995; revised and accepted February 13, 1996. * Supported by British Columbia Medical Services Foundation grant no. 92-58, Burnaby, British Columbia, Canada. t Presented at the 41st Annual Meeting ofthe Canadian Fertility and Andrology Society, Montebello, Quebec, Canada, September 20 to 23, 1995. :j: Reprint requests: Mary D. Stephenson, M.D., Department of Obstetrics and Gynaecology, 2nd floor Willow Pavilion, 855 West 12th Avenue, Vancouver, British Columbia V5Z 1M9, Canada (FAX: 604-875-3136).
24
Stephenson Factor frequencies in habitual abortion
recognized pregnancies (;;,:6 weeks' gestation) end in spontaneous abortion (3), approximately half of which are due to aneuploidy, most commonly, trisomy, monosomy, and polyploidy (4). Based on this figure, 0.3% of reproductive-aged couples should have a history of three consecutive losses, but epidemiological studies estimate that this history occurs more frequently, in the range of 1% to 2% (5). The observation of Poland et a1. (6) that couples with a history of three consecutive abortions, without an antecedent live birth, have a subsequent spontaneous abortion risk of 50%, strongly suggests that habitual abortion is a medical problem requiring investigation and management. In summary, it appears that a history of habitual abortion can occur randomly or can be related to some inherent factor that places the couple at risk for further pregnancy losses.
Fertility and Sterility®
r
Over the past few decades a number of factors have been proposed, initially based on retrospective studies. With improvements in clinical trial design and the development of immunologic evaluations in reproduction, modifications of previously reported diagnostic screening protocols are needed to reflect these scientific advances of the last decade. This paper presents the results of a prospective cohort study of 197 couples with a history of habitual abortion who were referred to the British Columbia Recurrent Pregnancy Loss Program, located in British Columbia's Women's Hospital and Health Centre, Vancouver, Canada. As with other provincial programs, transport assistance subsidies and overnight accommodation are available for patients having to travel to Vancouver. Physicians throughout the province of British Columbia (population of approximately 3.6 million) are encouraged to use this multidisciplinary program for the investigation and management of recurrent pregnancy loss. Because it is the only academic site in British Columbia for recurrent pregnancy loss research, patients from throughout the province are referred for evaluation. Stringent criteria were used to define factors associated with a history of habitual abortion. Couples were evaluated initially for genetic, endocrine, infectious, anatomical, and autoimmune factors. If the initial evaluation was negative, couples were classified as having unexplained habitual abortion and were subclassified as primary, secondary, or unclassified, based on the woman's partner-specific obstetric history. The diagnostic screening protocol and the results of this prospective cohort study can be used clinically to evaluate and counsel couples with a history of habitual abortion. MATERIALS AND METHODS
Between June 1992 and December 1994, all couples with a history of habitual abortion who were referred to the British Columbia Recurrent Pregnancy Loss Program were evaluated for genetic, endocrine, infectious, anatomical, and autoimmune factors. Habitual abortion was defined as three or more documented consecutive spontaneous pregnancy losses less than 20 weeks' gestation, excluding any spontaneous abortions with documented aneuploidy by karyotype analysis. Previous pregnancies were considered documented if a urinary or serum lJ-hCG was positive or a gestational sac was seen on ultrasound. If an ectopic pregnancy was intermixed between the consecutive spontaneous abortions, it was excluded. Structural genetic factors, such as balanced Robertsonian or reciprocal translocations, were identi-
Vol. 66, No.1, July 1996
fied with karotype analysis of both partners, using peripheral blood lymphocyte cultures and Giemsa banding. Endocrine factors were evaluated with serum TSH and PRL assays and late luteal phase endometrial biopsies. A luteal phase deficiency was defined as two late luteal phase endometrial biopsies with maturation delays of :=:=3 days, based on the first day of the next menstrual period. Endometrial dating was based on the criteria of Noyes et al. (7). An infectious factor, specifically endometritis, which was defined as persistent plasma cells in two endometrial biopsies (8), was evaluated concomitantly with the evaluation of the luteal phase. Cultures of the endometrium were taken from the second endometrial biopsy if plasma cells were present in the first, so that appropriate antibiotic therapy could be instituted. Anatomical factors, including severe intrauterine adhesions and miillerian tract anomalies, were defined according to the American Fertility Society classification (9). An arcuate uterus or mild to moderate intrauterine adhesions were considered to be of no significance. The uterine cavity was evaluated by standard roentgenogram hysterosalpingography (HSG) or office hysteroscopy. If the uterine contour was suggestive of either a septate or a bicornuate uterus, hysterosonography or a diagnostic laparoscopy was performed. The diagnosis of cervical incompetence was made when there was a history of a second trimester loss, the HSG revealed an internal os diameter of 10 mm, and, at hysteroscopy, there was no resistance with insertion of no. 17 Pratt dilator. Autoimmune factors that were evaluated included the anti phospholipid antibody syndrome and the undifferentiated connective tissue syndrome. The antiphospholipid antibody syndrome was defined as persistent (on repeat testing :=:=6 weeks apart) elevation ofthe lupus anticoagulant and/or anticardiolipin immunoglobulins G or M. The undifferentiated connective tissue syndrome was defined as persistent elevation of the antinuclear antibody titer (:=:=1:160) and clinical features suggestive of, but not meeting the diagnostic criteria of, other recognized systemic rheumatic diseases (10). The presence of the lupus anticoagulant was defined by the persistent prolongation of the activated partial thromboplastin time, which did not correct with the addition of normal plasma, or, persistent prolongation of the dilute Russel viper venom test ratio, which corrected with the addition of platelet phospholipid (11). Anticardiolipin immunoglobulin G and M levels were determined using ELISAs, which were standardized as defined by the 1986
Stephenson Factor frequencies in habitual abortion
25
International Workshop on the evaluation of the anticardiolipin test (12). If the couple's evaluation was nonnal, they were classified as having unexplained habitual abortion. Subclassification of this unexplained group was based on the woman's partner-specific obstetric history. Primary unexplained habitual abortion was defined as habitual abortion without an antecedent pregnancy beyond 20 weeks' gestation, with the woman's present reproductive partner. Secondary unexplained habitual abortion was defined as habitual abortion with one or more antecedent pregnancy(ies) beyond 20 weeks' gestation, with the woman's present reproductive partner. Unclassified unexplained habitual abortion was used for couples who did not fit strictly into either the primary or secondary subclassification, predominantly because of more than one reproductive partner in the woman's history of habitual abortion. A Recurrent Pregnancy Loss Database was created using ACCESS 2.0 (Microsoft, Redmond, WA). Partner-specific obstetric histories, personal data, and test results from all couples who were evaluated in this Program were entered into the database. The frequency of genetic, endocrine, infectious, anatomical, and autoimmune factors associated with a history of habitual abortion was obtained from this data.
RESULTS Between June 1992 and December 1994, 284 couples with a history of three or more documented spontaneous abortions were seen in the British Columbia Recurrent Pregnancy Loss Program. Thirtyseven couples were excluded because their spontaneous abortions were not consecutive. Of the 247 couples remaining, 17 were not interested in being investigated or were lost to follow-up. A further 33 couples were excluded because of documented aneuploidy in one or more of their spontaneous abortions, reducing their number of consecutive spontaneous abortions below three. This left a cohort of 197 cou-
Table 1 Frequency of Factors Associated With Habitual Abortion in 197 Couples Factor
No. of couples
Frequency %
Genetic (structural) Endocrine Infectious Anatomical Autoimmune Unexplained
26
7 39 1 31 40 84
3.5 20 0.5 16 20 43
Stephenson Factor frequencies in habitual abortion
Table 2 Structural Karyotypic Abnormalities in Couples With Habitual Abortion (n = 7) 46,XX, 46,XY, 46,xx, 45,XX, 46,XY, 46,XX, 45,XX,
inversion 6 (pI2.2p25.1) insertion 17 (q23pI2pI3) translocation (4;6)(q31.3;q21) translocation (13q22q) translocation (7;13)(pI3;q21.2) translocation (11;21) translocation (13;14)
pIes with a history of habitual abortion to be evaluated. The mean age of the female partner was 33 years (range 21 to 46 years). There were 843 spontaneous abortions documented in the 197 obstetric histories. The mean number of documented consecutive spontaneous abortions in this cohort was 4.1 (range 3 to 12). The factors associated with their histories of habitual abortion are shown in Table 1. A structural genetic factor was identified in 3.5% (7/197) of the couples, of which five were maternal and two were paternal. The structural abnonnalities are listed in Table 2. An endocrine factor was identified in 20% (39/197) of the women. Of the 39 women, 34 were found to have a luteal phase deficiency and 6 were found to be hypothyroid. An infectious factor was identified in one woman; the cultures were negative and therefore she was treated for nonspecific endometritis. An anatomical factor was identified in 16% (311 197) of the women. Of the 31 women, 15 were found to have mtillerian tract anomalies; 8 had septate uteri, 4 had T -shaped uteri, 1 had a bicornuate uterus, 1 had an uterus didelphys, and 1 had an unicornuate uterus with a rudimentary horn. Severe intrauterine adhesions were documented in 11 women, 4 were found to have cervical incompetence and 1 woman was found to have a submucous fibroid. An autoimmune factor was identified in 20% (401 197) of the women. Thirty-four were classified as having the antiphospholipid antibody syndrome, and six were classified as having the undifferentiated connective tissue syndrome. It was interesting to note that many of the women with either the antiphospholipid antibody syndrome or the undifferentiated connective tissue syndrome had clinical and/or laboratory features of other autoimmune disorders, including Hashimoto's thyroiditis, undifferentiated oligoarthritis with post-Rubella immunization arthritis, autoimmune liver disease, and autoimmune neutropenia. Eighty-four couples who completed the diagnostic screening protocol were classified as unexplained habitual abortion because genetic, endocrine, infectious, anatomical, and autoimmune factors were not
Fertility and Sterility®
identified. Of these 84 couples, 55 (65%) were subclassified as primary, 23 (27%) were subclassified as secondary, and 6 (7%) had unclassified unexplained habitual abortion. DISCUSSION
With the establishment of the British Columbia Recurrent Pregnancy Loss Program in June 1992, a unique opportunity was created to study prospectively the frequency of factors associated with a history of habitual abortion in a large population cohort. In recent reviews, there is general agreement in regard to genetic, endocrine, anatomical, and autoimmune factors being associated with a history of habitual abortion (1, 14, 15). The diagnostic screening protocol in this paper includes not only the standard methods of evaluation for these factors, but also uses some of the newer technologies available. Office hysteroscopy was used instead of roentgenogram HSG when there was no concomitant infertility. When it was necessary to evaluate the external contour of the uterus and there was no concomitant infertility, hysterosonography was offered as an alternative to diagnostic laparoscopy. Alternatively, in these recent reviews, there is considerable disagreement in regard to other potential factors associated with habitual abortion, such as infectious, alloimmune, psychological, and environmental factors. The diagnostic screening protocol in this paper did include evaluation of an infectious factor. The diagnosis of chronic endometritis was made by persistent identification of plasma cells in the luteal phase endometrial biopsies. Because a luteal phase biopsy of a chronic endometrial infection with either Mycoplasma hominis or Ureaplasma urealyticum would be characterized by an inflammatory response including plasma cells; endometrial biopsies, rather than cultures of the endometrium, which are prone to cervical contamination, were chosen as the screening test for an infectious factor. Couples classified as having unexplained habitual abortion were offered alloimmune testing, but they were not classified as such because there is little agreement in the scientific literature with regard to the definition of an alloimmune factor. The alloimmune testing consisted of human leukocyte antigen typing of both partners, the mixed lymphocyte culture, and assessment of maternal immunoglobulins G and M directed against paternal T and B lymphocytes, using both the complement-dependent cytotoxicity assay and the complement-independent flow cytometric crossmatch (13). A study comparing alloimmune testing in couples with unexplained habitual abortion and matched controls is in progress. The results of this study will be forthcoming.
Vol. 66, No.1, July 1996
In prior publications outlining diagnostic screening protocols, some of the present factors associated with habitual abortion were not evaluated. Therefore, frequency of factor comparisons between older publications and this paper are of limited significance. Historically, Stray-Pedersen and Stray-Pedersen (16) published the first prospective cohort of couples (n = 195) with a history of three or more documented consecutive spontaneous abortions. They identified 3% of couples with a structural genetic factor, 5% with an endocrine factor, 15% with an endometrial infection, 28% with an anatomical factor, and 4% with a sperm factor, leaving 44% with unknown etiology. The evaluation was dissimilar to the present study in many aspects. A luteal phase deficiency was diagnosed by a short luteal phase, low P levels, and a single abnormal endometrial biopsy. The high percentage of women classified as having an endometrial infections, defined as positive cervical mucous or endometrium cultures, could be explained by vaginal and cervical contamination of the specimens for culture. Anatomical factors were evaluated using only hysterography and cervical insertion of Hegar dilators. Cervical incompetence was the most common type of anatomical factor, accounting for almost half of this anatomical group. Fixed retroversion and the presence of uterine fibroids also were included as significant findings. In the present study, cervical incompetence was defined more strictly and only fibroids found to be submucous in location were included as being significant. The major difference between the diagnostic screening protocol of Stray-Pedersen and StrayPedersen (16) and the present study is that the former did not include an autoimmune evaluation. With the subsequent development of sensitive clotting-based and ELISAs in the 1980s (11, 12), came the ability to identify women with the antiphospholipid antibody syndrome. As in this present study, alloimmune testing was mentioned but not reported. More recently, Makino et al. (17) published a survey of 1,120 Japanese women with repeated spontaneous abortions. However, they included women with two or more spontaneous abortions, which were not necessarily consecutive. With this liberal entry criteria, it was more likely that many ofthe spontaneous abortions had occurred randomly. As well, the evaluations were not offered universally to all women in the survey. Makino et al. (17) primarily focused on the evaluation of congenital uterine anomalies using a new HSG technique that was performed on 1,000 of the women. Fifteen percent ofthe women were identified as having congenital uterine anomalies, although 65% were arcuate uteri, which was not considered
Stephenson Factor frequencies in habitual abortion
27
significant in the present study. Only 148 of 1,120 women were evaluated for an autoimmune factor. Anticardiolipin immunoglobulins were used as the only diagnostic test for the antiphospholipid antibody syndrome and 16% of the women tested had elevated results. Although it remains the largest study in the literature to date, the many weaknesses in the study design do not allow it to be classified as the definitive study on frequency of factors associated with habitual abortion. Tulppala et al. published a comprehensive evaluation of genetic, endocrine, infectious, anatomical, and autoimmune factors associated with recurrent spontaneous abortion (18). Although their sample size was limited to 65 patients, the investigative protocol was similar to the current study. The population cohort was slightly different, as 34 of the women had three consecutive spontaneous abortions and 29 had four to eight spontaneous abortions that were not necessarily consecutive. Their data were similar to the current study for genetic, endocrine, infectious, and anatomical factors. However, an autoimmune factor was identified in only 6 of 65 patients (9%) compared with 20% in the current study, although the autoimmune evaluation of Tulppala et al. (18) consisted of only ELISAs for anticardiolipin immunoglobulins whereas the present study also included clotting-based assays to assess for the presence of the lupus anticoagulant. In this current study, 33 couples were excluded because of documented aneuploidy in one or more abortions, which reduced their number of consecutive spontaneous abortions without aneuploidy to below three. The present disregard for aneuploidy in the standard definition of habitual abortion may be contributing to the difficulty of evaluating treatment efficacy in couples with factor associated habitual abortion. In addition, although aneuploidy is generally accepted to occur in approximately half of all spontaneous abortions, the risk of aneuploidy also increases with advancing maternal age (5). Because women with a history of habitual abortion generally are older, as confirmed in this study, a substantial number of their spontaneous abortions could have been attributable to aneuploidy, ifkaryotype analysis was performed more frequently. Because pregnancy loss is such an emotional event, documentation of aneuploidy could help patients understand why it occurred and subsequently help them to overcome some of their grief associated with the loss and their anxiety associated with the anticipation of another conception. Excluding spontaneous abortions with documented aneuploidy from the total number of consecutive spontaneous abortions would help to prevent unnecessary and costly
28
Stephenson Factor frequencies in habitual abortion
investigations and treatments, which also are challenging psychologically to patients. As well as the emotional benefit, Wolf and Horger (19) recently calculated a significant financial benefit to the health care system if a policy of karyotype analysis of recurrent abortion specimens was recommended. Documentation of aneuploidy therefore is important, both psychologically to the patients and to improve the accuracy of the diagnostic process. In conclusion, this prospective cohort study has identified genetic, endocrine, infectious, anatomical, and autoimmune factors associated with a history of habitual abortion in approximately 60% of the couples evaluated. Pregnancy outcome data after treatment ofthe factors will be addressed in a subsequent paper.
Acknowledgments. I thank Cathe Marshall, C.C.H.R.A.(C.), British Columbia's Women's Hospital and Health Centre, Vancouver, Canada, for the data preparation and Margo R. Fluker, M.D., University of British Columbia, Vancouver, Canada, for her assistance with the manuscript.
REFERENCES 1. Stirrat GM. Recurrent spontaneous abortion. In: Coulam CB, Faulk WP, McIntyre JA, editors. Immunological obstetrics. New York: Norton, 1992:357-76. 2. Hook EB, Porter IH. Terminological conventions, methodological considerations, temporal trends, specific genes, environmental hazards and some other factors pertaining to embryonic and fetal deaths. In: Porter IH, Hook EB, editors. Human embryonic and fetal death. London: Academic Press, 1980:1-18. 3. Kline J, Stein Z. Very early pregnancy. In: Dixon RL, editor. Reproductive toxicology. New York: Raven, 1985:251-65. 4. Jacobs PA, Hassold T. Chromosome abnormalities: origin and etiology in abortions and livebirths. In: Vogel F, Sperling K, editors. Human genetics. Berlin: Springer-Verlag, 1987: 233-44. 5. Roman E. Fetal loss rates and their relation to pregnancy order. J Epidemiol Community Health 1984;38:29-35. 6. Poland BJ, Miller JR, Jones DC, Trimple BK. Reproductive counselling in patients who have a spontaneous abortion. Am J Obstet Gynecol 1977; 127:685-91. 7. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3-25. 8. Kurman RJ, Mazur MT. Benign diseases of the endometrium. In: Kurman R, editor. Blaustein's pathology of the female genital tract, 3rd ed. New York: Springer-Verlag, 1987: 295-6. 9. The American Fertility Society. The American Fertility Society classification of adnexal adhesions, distal tubal occlusion, tubal occlusion secondary to tubal ligation, tubal pregnancies, mullerian anomalies and intrauterine adhesions. Fertil Steril 1988;49:944-55. 10. Reichlin M. Undifferentiated connective tissue syndromes. In: Schumacher HR Jr, Klippel J, Koopman IU, editors. The
Fertility and Sterility®
11.
12.
13.
14. 15.
primer on the rheumatic disease, 10th ed. Atlanta: Arthritis Foundation, 1993:135-6. Triplett DA, Brandt J, Kaczor D, Schaeffer J. Laboratory diagnosis of lupus inhibitors: a comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure. Am J Clin Pathol 1983; 79:678-82. Harris EN, Gharavi AE, Patel SS, Hughes GRV. Evaluation of the anticardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987;68: 215-22. Stephenson MD, Wu V, Mackinnon M, Sadoway J, Keown P. Standardization of flow cytometric crossmatch (FCXM) for investigation of unexplained habitual abortion. Am J Reprod Immunol1995;33:1-9. American College of Obstetrics and Gynaecology. Early pregnancy loss. ACOG Tech Bull 1995;212:1-7. SperoffL, Glass R, Kase N. Recurrent early pregnancy losses.
Vol. 66, No.1, July 1996
16.
17.
18.
19.
In: Clinical gynaecologic endocrinology and infertility. 5th ed. Baltimore: Williams & Wilkins, 1994:841-51. Stray-Pedersen B, Stray-Pedersen S. Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984; 148:140-6. Makino T, Hara T, Oka C, Toyoshima K, Sugi T, Iwasaki K, et al. Survey of 1120 Japanese women with a history of recurrent spontaneous abortions. Eur J Obstet Gynecol Reprod BioI 1992;44:123-30. Tulppala M, Palosuo T, Ramsay T, Miettinen A, Salonen R, Ylikorkala O. A prospective study of63 couples with a history of recurrent spontaneous abortion: contributing factors and outcome of subsequent pregnancies. Hum Reprod 1993; 8:764-77. Wolf GC, Horger EO. Indications for examination of spontaneous abortion specimens: a reassessment. Am J Obstet Gynecol 1995; 173:1364-8.
Stephenson Factor frequencies in habitual abortion
29
,t'J
Vol. 66, No.1, July 1996 Printed on acidfree paper in U. S. A.
1996 American Society for Reproductive Medicine
Frequency of factors associated with habitual abortion in 197 couples*t
Mary D. Stephenson, M.D.:!: Department of Obstetrics and Gynaecology, University of British Columbia, British Columbia's Women's Hospital and Health Centre, Vancouver, British Columbia, Canada
Objective: To determine the frequency of factors associated with habitual abortion in 197 couples. Design: Prospective cohort study. Setting: The British Columbia Recurrent Pregnancy Loss Program, located in a tertiary care academic center. Interventions: Diagnostic screening protocol. Main Outcome Measures: Genetic, endocrine, infectious, anatomical, and autoimmune factors associated with habitual abortion. Results: A structural genetic factor was identified in 3.5% ofthe couples. An endocrine factor, including luteal phase deficiency and hypothyroidism, was identified in 20% and an infectious factor was identified in 0.5% of the couples. An anatomical factor, including various miillerian tract anomalies and severe intrauterine adhesions, was found in 16% and an autoimmune factor, including the antiphospholipid antibody syndrome and the undifferentiated connective tissue syndrome, was identified in 20% of the couples. Eighty-four couples who completed the diagnostic screening protocol were classified as having unexplained habitual abortion. Of these 84 couples, 65% were subclassified as primary, 27% were subclassified as secondary, and 7% had unclassified unexplained habitual abortion. Conclusions: This large-scale study identified genetic, endocrine, infectious, anatomical, or autoimmune factors in approximately 60% of couples with habitual abortion. Fertil Steril® 1996;66:24-9 Key Words: Habitual abortion, etiology, factors, frequency
Habitual abortion previously has been defined as three or more consecutive spontaneous abortions (1). The World Health Organization has defined an abortion as "the expulsion or extraction from its mother of an embryo or fetus weighing 500 grams or less" (2), which corresponds to a gestational age between 20 and 22 weeks. It appears that 15% of all clinically Received October 18, 1995; revised and accepted February 13, 1996. * Supported by British Columbia Medical Services Foundation grant no. 92-58, Burnaby, British Columbia, Canada. t Presented at the 41st Annual Meeting ofthe Canadian Fertility and Andrology Society, Montebello, Quebec, Canada, September 20 to 23, 1995. :j: Reprint requests: Mary D. Stephenson, M.D., Department of Obstetrics and Gynaecology, 2nd floor Willow Pavilion, 855 West 12th Avenue, Vancouver, British Columbia V5Z 1M9, Canada (FAX: 604-875-3136).
24
Stephenson Factor frequencies in habitual abortion
recognized pregnancies (;;,:6 weeks' gestation) end in spontaneous abortion (3), approximately half of which are due to aneuploidy, most commonly, trisomy, monosomy, and polyploidy (4). Based on this figure, 0.3% of reproductive-aged couples should have a history of three consecutive losses, but epidemiological studies estimate that this history occurs more frequently, in the range of 1% to 2% (5). The observation of Poland et a1. (6) that couples with a history of three consecutive abortions, without an antecedent live birth, have a subsequent spontaneous abortion risk of 50%, strongly suggests that habitual abortion is a medical problem requiring investigation and management. In summary, it appears that a history of habitual abortion can occur randomly or can be related to some inherent factor that places the couple at risk for further pregnancy losses.
Fertility and Sterility®
r
Over the past few decades a number of factors have been proposed, initially based on retrospective studies. With improvements in clinical trial design and the development of immunologic evaluations in reproduction, modifications of previously reported diagnostic screening protocols are needed to reflect these scientific advances of the last decade. This paper presents the results of a prospective cohort study of 197 couples with a history of habitual abortion who were referred to the British Columbia Recurrent Pregnancy Loss Program, located in British Columbia's Women's Hospital and Health Centre, Vancouver, Canada. As with other provincial programs, transport assistance subsidies and overnight accommodation are available for patients having to travel to Vancouver. Physicians throughout the province of British Columbia (population of approximately 3.6 million) are encouraged to use this multidisciplinary program for the investigation and management of recurrent pregnancy loss. Because it is the only academic site in British Columbia for recurrent pregnancy loss research, patients from throughout the province are referred for evaluation. Stringent criteria were used to define factors associated with a history of habitual abortion. Couples were evaluated initially for genetic, endocrine, infectious, anatomical, and autoimmune factors. If the initial evaluation was negative, couples were classified as having unexplained habitual abortion and were subclassified as primary, secondary, or unclassified, based on the woman's partner-specific obstetric history. The diagnostic screening protocol and the results of this prospective cohort study can be used clinically to evaluate and counsel couples with a history of habitual abortion. MATERIALS AND METHODS
Between June 1992 and December 1994, all couples with a history of habitual abortion who were referred to the British Columbia Recurrent Pregnancy Loss Program were evaluated for genetic, endocrine, infectious, anatomical, and autoimmune factors. Habitual abortion was defined as three or more documented consecutive spontaneous pregnancy losses less than 20 weeks' gestation, excluding any spontaneous abortions with documented aneuploidy by karyotype analysis. Previous pregnancies were considered documented if a urinary or serum lJ-hCG was positive or a gestational sac was seen on ultrasound. If an ectopic pregnancy was intermixed between the consecutive spontaneous abortions, it was excluded. Structural genetic factors, such as balanced Robertsonian or reciprocal translocations, were identi-
Vol. 66, No.1, July 1996
fied with karotype analysis of both partners, using peripheral blood lymphocyte cultures and Giemsa banding. Endocrine factors were evaluated with serum TSH and PRL assays and late luteal phase endometrial biopsies. A luteal phase deficiency was defined as two late luteal phase endometrial biopsies with maturation delays of :=:=3 days, based on the first day of the next menstrual period. Endometrial dating was based on the criteria of Noyes et al. (7). An infectious factor, specifically endometritis, which was defined as persistent plasma cells in two endometrial biopsies (8), was evaluated concomitantly with the evaluation of the luteal phase. Cultures of the endometrium were taken from the second endometrial biopsy if plasma cells were present in the first, so that appropriate antibiotic therapy could be instituted. Anatomical factors, including severe intrauterine adhesions and miillerian tract anomalies, were defined according to the American Fertility Society classification (9). An arcuate uterus or mild to moderate intrauterine adhesions were considered to be of no significance. The uterine cavity was evaluated by standard roentgenogram hysterosalpingography (HSG) or office hysteroscopy. If the uterine contour was suggestive of either a septate or a bicornuate uterus, hysterosonography or a diagnostic laparoscopy was performed. The diagnosis of cervical incompetence was made when there was a history of a second trimester loss, the HSG revealed an internal os diameter of 10 mm, and, at hysteroscopy, there was no resistance with insertion of no. 17 Pratt dilator. Autoimmune factors that were evaluated included the anti phospholipid antibody syndrome and the undifferentiated connective tissue syndrome. The antiphospholipid antibody syndrome was defined as persistent (on repeat testing :=:=6 weeks apart) elevation ofthe lupus anticoagulant and/or anticardiolipin immunoglobulins G or M. The undifferentiated connective tissue syndrome was defined as persistent elevation of the antinuclear antibody titer (:=:=1:160) and clinical features suggestive of, but not meeting the diagnostic criteria of, other recognized systemic rheumatic diseases (10). The presence of the lupus anticoagulant was defined by the persistent prolongation of the activated partial thromboplastin time, which did not correct with the addition of normal plasma, or, persistent prolongation of the dilute Russel viper venom test ratio, which corrected with the addition of platelet phospholipid (11). Anticardiolipin immunoglobulin G and M levels were determined using ELISAs, which were standardized as defined by the 1986
Stephenson Factor frequencies in habitual abortion
25
International Workshop on the evaluation of the anticardiolipin test (12). If the couple's evaluation was nonnal, they were classified as having unexplained habitual abortion. Subclassification of this unexplained group was based on the woman's partner-specific obstetric history. Primary unexplained habitual abortion was defined as habitual abortion without an antecedent pregnancy beyond 20 weeks' gestation, with the woman's present reproductive partner. Secondary unexplained habitual abortion was defined as habitual abortion with one or more antecedent pregnancy(ies) beyond 20 weeks' gestation, with the woman's present reproductive partner. Unclassified unexplained habitual abortion was used for couples who did not fit strictly into either the primary or secondary subclassification, predominantly because of more than one reproductive partner in the woman's history of habitual abortion. A Recurrent Pregnancy Loss Database was created using ACCESS 2.0 (Microsoft, Redmond, WA). Partner-specific obstetric histories, personal data, and test results from all couples who were evaluated in this Program were entered into the database. The frequency of genetic, endocrine, infectious, anatomical, and autoimmune factors associated with a history of habitual abortion was obtained from this data.
RESULTS Between June 1992 and December 1994, 284 couples with a history of three or more documented spontaneous abortions were seen in the British Columbia Recurrent Pregnancy Loss Program. Thirtyseven couples were excluded because their spontaneous abortions were not consecutive. Of the 247 couples remaining, 17 were not interested in being investigated or were lost to follow-up. A further 33 couples were excluded because of documented aneuploidy in one or more of their spontaneous abortions, reducing their number of consecutive spontaneous abortions below three. This left a cohort of 197 cou-
Table 1 Frequency of Factors Associated With Habitual Abortion in 197 Couples Factor
No. of couples
Frequency %
Genetic (structural) Endocrine Infectious Anatomical Autoimmune Unexplained
26
7 39 1 31 40 84
3.5 20 0.5 16 20 43
Stephenson Factor frequencies in habitual abortion
Table 2 Structural Karyotypic Abnormalities in Couples With Habitual Abortion (n = 7) 46,XX, 46,XY, 46,xx, 45,XX, 46,XY, 46,XX, 45,XX,
inversion 6 (pI2.2p25.1) insertion 17 (q23pI2pI3) translocation (4;6)(q31.3;q21) translocation (13q22q) translocation (7;13)(pI3;q21.2) translocation (11;21) translocation (13;14)
pIes with a history of habitual abortion to be evaluated. The mean age of the female partner was 33 years (range 21 to 46 years). There were 843 spontaneous abortions documented in the 197 obstetric histories. The mean number of documented consecutive spontaneous abortions in this cohort was 4.1 (range 3 to 12). The factors associated with their histories of habitual abortion are shown in Table 1. A structural genetic factor was identified in 3.5% (7/197) of the couples, of which five were maternal and two were paternal. The structural abnonnalities are listed in Table 2. An endocrine factor was identified in 20% (39/197) of the women. Of the 39 women, 34 were found to have a luteal phase deficiency and 6 were found to be hypothyroid. An infectious factor was identified in one woman; the cultures were negative and therefore she was treated for nonspecific endometritis. An anatomical factor was identified in 16% (311 197) of the women. Of the 31 women, 15 were found to have mtillerian tract anomalies; 8 had septate uteri, 4 had T -shaped uteri, 1 had a bicornuate uterus, 1 had an uterus didelphys, and 1 had an unicornuate uterus with a rudimentary horn. Severe intrauterine adhesions were documented in 11 women, 4 were found to have cervical incompetence and 1 woman was found to have a submucous fibroid. An autoimmune factor was identified in 20% (401 197) of the women. Thirty-four were classified as having the antiphospholipid antibody syndrome, and six were classified as having the undifferentiated connective tissue syndrome. It was interesting to note that many of the women with either the antiphospholipid antibody syndrome or the undifferentiated connective tissue syndrome had clinical and/or laboratory features of other autoimmune disorders, including Hashimoto's thyroiditis, undifferentiated oligoarthritis with post-Rubella immunization arthritis, autoimmune liver disease, and autoimmune neutropenia. Eighty-four couples who completed the diagnostic screening protocol were classified as unexplained habitual abortion because genetic, endocrine, infectious, anatomical, and autoimmune factors were not
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identified. Of these 84 couples, 55 (65%) were subclassified as primary, 23 (27%) were subclassified as secondary, and 6 (7%) had unclassified unexplained habitual abortion. DISCUSSION
With the establishment of the British Columbia Recurrent Pregnancy Loss Program in June 1992, a unique opportunity was created to study prospectively the frequency of factors associated with a history of habitual abortion in a large population cohort. In recent reviews, there is general agreement in regard to genetic, endocrine, anatomical, and autoimmune factors being associated with a history of habitual abortion (1, 14, 15). The diagnostic screening protocol in this paper includes not only the standard methods of evaluation for these factors, but also uses some of the newer technologies available. Office hysteroscopy was used instead of roentgenogram HSG when there was no concomitant infertility. When it was necessary to evaluate the external contour of the uterus and there was no concomitant infertility, hysterosonography was offered as an alternative to diagnostic laparoscopy. Alternatively, in these recent reviews, there is considerable disagreement in regard to other potential factors associated with habitual abortion, such as infectious, alloimmune, psychological, and environmental factors. The diagnostic screening protocol in this paper did include evaluation of an infectious factor. The diagnosis of chronic endometritis was made by persistent identification of plasma cells in the luteal phase endometrial biopsies. Because a luteal phase biopsy of a chronic endometrial infection with either Mycoplasma hominis or Ureaplasma urealyticum would be characterized by an inflammatory response including plasma cells; endometrial biopsies, rather than cultures of the endometrium, which are prone to cervical contamination, were chosen as the screening test for an infectious factor. Couples classified as having unexplained habitual abortion were offered alloimmune testing, but they were not classified as such because there is little agreement in the scientific literature with regard to the definition of an alloimmune factor. The alloimmune testing consisted of human leukocyte antigen typing of both partners, the mixed lymphocyte culture, and assessment of maternal immunoglobulins G and M directed against paternal T and B lymphocytes, using both the complement-dependent cytotoxicity assay and the complement-independent flow cytometric crossmatch (13). A study comparing alloimmune testing in couples with unexplained habitual abortion and matched controls is in progress. The results of this study will be forthcoming.
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In prior publications outlining diagnostic screening protocols, some of the present factors associated with habitual abortion were not evaluated. Therefore, frequency of factor comparisons between older publications and this paper are of limited significance. Historically, Stray-Pedersen and Stray-Pedersen (16) published the first prospective cohort of couples (n = 195) with a history of three or more documented consecutive spontaneous abortions. They identified 3% of couples with a structural genetic factor, 5% with an endocrine factor, 15% with an endometrial infection, 28% with an anatomical factor, and 4% with a sperm factor, leaving 44% with unknown etiology. The evaluation was dissimilar to the present study in many aspects. A luteal phase deficiency was diagnosed by a short luteal phase, low P levels, and a single abnormal endometrial biopsy. The high percentage of women classified as having an endometrial infections, defined as positive cervical mucous or endometrium cultures, could be explained by vaginal and cervical contamination of the specimens for culture. Anatomical factors were evaluated using only hysterography and cervical insertion of Hegar dilators. Cervical incompetence was the most common type of anatomical factor, accounting for almost half of this anatomical group. Fixed retroversion and the presence of uterine fibroids also were included as significant findings. In the present study, cervical incompetence was defined more strictly and only fibroids found to be submucous in location were included as being significant. The major difference between the diagnostic screening protocol of Stray-Pedersen and StrayPedersen (16) and the present study is that the former did not include an autoimmune evaluation. With the subsequent development of sensitive clotting-based and ELISAs in the 1980s (11, 12), came the ability to identify women with the antiphospholipid antibody syndrome. As in this present study, alloimmune testing was mentioned but not reported. More recently, Makino et al. (17) published a survey of 1,120 Japanese women with repeated spontaneous abortions. However, they included women with two or more spontaneous abortions, which were not necessarily consecutive. With this liberal entry criteria, it was more likely that many ofthe spontaneous abortions had occurred randomly. As well, the evaluations were not offered universally to all women in the survey. Makino et al. (17) primarily focused on the evaluation of congenital uterine anomalies using a new HSG technique that was performed on 1,000 of the women. Fifteen percent ofthe women were identified as having congenital uterine anomalies, although 65% were arcuate uteri, which was not considered
Stephenson Factor frequencies in habitual abortion
27
significant in the present study. Only 148 of 1,120 women were evaluated for an autoimmune factor. Anticardiolipin immunoglobulins were used as the only diagnostic test for the antiphospholipid antibody syndrome and 16% of the women tested had elevated results. Although it remains the largest study in the literature to date, the many weaknesses in the study design do not allow it to be classified as the definitive study on frequency of factors associated with habitual abortion. Tulppala et al. published a comprehensive evaluation of genetic, endocrine, infectious, anatomical, and autoimmune factors associated with recurrent spontaneous abortion (18). Although their sample size was limited to 65 patients, the investigative protocol was similar to the current study. The population cohort was slightly different, as 34 of the women had three consecutive spontaneous abortions and 29 had four to eight spontaneous abortions that were not necessarily consecutive. Their data were similar to the current study for genetic, endocrine, infectious, and anatomical factors. However, an autoimmune factor was identified in only 6 of 65 patients (9%) compared with 20% in the current study, although the autoimmune evaluation of Tulppala et al. (18) consisted of only ELISAs for anticardiolipin immunoglobulins whereas the present study also included clotting-based assays to assess for the presence of the lupus anticoagulant. In this current study, 33 couples were excluded because of documented aneuploidy in one or more abortions, which reduced their number of consecutive spontaneous abortions without aneuploidy to below three. The present disregard for aneuploidy in the standard definition of habitual abortion may be contributing to the difficulty of evaluating treatment efficacy in couples with factor associated habitual abortion. In addition, although aneuploidy is generally accepted to occur in approximately half of all spontaneous abortions, the risk of aneuploidy also increases with advancing maternal age (5). Because women with a history of habitual abortion generally are older, as confirmed in this study, a substantial number of their spontaneous abortions could have been attributable to aneuploidy, ifkaryotype analysis was performed more frequently. Because pregnancy loss is such an emotional event, documentation of aneuploidy could help patients understand why it occurred and subsequently help them to overcome some of their grief associated with the loss and their anxiety associated with the anticipation of another conception. Excluding spontaneous abortions with documented aneuploidy from the total number of consecutive spontaneous abortions would help to prevent unnecessary and costly
28
Stephenson Factor frequencies in habitual abortion
investigations and treatments, which also are challenging psychologically to patients. As well as the emotional benefit, Wolf and Horger (19) recently calculated a significant financial benefit to the health care system if a policy of karyotype analysis of recurrent abortion specimens was recommended. Documentation of aneuploidy therefore is important, both psychologically to the patients and to improve the accuracy of the diagnostic process. In conclusion, this prospective cohort study has identified genetic, endocrine, infectious, anatomical, and autoimmune factors associated with a history of habitual abortion in approximately 60% of the couples evaluated. Pregnancy outcome data after treatment ofthe factors will be addressed in a subsequent paper.
Acknowledgments. I thank Cathe Marshall, C.C.H.R.A.(C.), British Columbia's Women's Hospital and Health Centre, Vancouver, Canada, for the data preparation and Margo R. Fluker, M.D., University of British Columbia, Vancouver, Canada, for her assistance with the manuscript.
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